Characterization of gyrA and parC mutations in ciprofloxacin-resistant Pseudomonas aeruginosa isolates from Tehran hospitals in Iran.

Background and Objectives
Pseudomonas aeruginosa, a major cause of several infectious diseases, has become a hazardous resistant pathogen. One of the factors contributing to quinolone resistance in P. aeruginosa is mutations occurring in gyrA and parC genes encoding the A subunits of type II and IV topoisomerases, respectively, in quinolone resistance determining regions (QRDR) of the bacterial chromosome.


Materials and Methods
Thirty seven isolates from patients with burn wounds and 20 isolates from blood, urine and sputum specimen were collected. Minimum Inhibitory Concentrations (MICs) of ciprofloxacin were determined by agar diffusion assay. Subsequently, QRDRs regions of gyrA and parC were amplified from resistant isolates and were assessed for mutations involved in ciprofloxacin resistance after sequencing.


Results
Nine isolates with MIC≥8 μg/ml had a mutation in gyrA (Thr83→Ile). Amongst these, seven isolates also had a mutation in parC (Ser87→ Leu or Trp) indicating that the prevalent mutation in gyrA is Thr83Ile and Ser87Leu/Trp in parC. No single parC mutation was observed.


Conclusion
It seems that mutations in gyrA are concomitant with mutations in parC which might lead to high-level ciprofloxacin resistance in P. aeruginosa isolates from patients with burn wounds and urinary tract infections.


INTRODUCTION
A growing population of multidrug resistant bacteria has emerged as the "post antibiotic era" of infectious diseases. One prominent example is Pseudomonas aeruginosa which has been the focus of therapeutic challenges. This ubiquitous organism exists in many diverse environments, and can be isolated from various living sources. The ability of P. aeruginosa to survive in harsh conditions and endure stress has allowed the organism to persist in both community and hospitals. P. aeruginosa is commonly responsible for nosocomial infections in ICUs, including surgical site, urinary tract, pneumonia and bloodstream, eye, ear, nose and throat infections (1)(2)(3)(4).
Due to the low permeability of its cell wall, P. aeruginosa is intrinsically resistant to most antibiotics as a result of decreased intracellular drug concentration caused by decreased uptake or increased efflux pumps expression (5)(6).
Principal mechanisms of bacterial resistance to quinolones are modification of the target site (DNA gyrase) and reduction of intracellular concentration of quinolones due to mutations in the regulatory genes mexR and nfxB (7). In P. aeruginosa, resistance to quinolones is also often mediated by mutations in regulatory genes leading to upregulation of different efflux pumps systems (8)(9)(10). Mutations in plasmid-mediated quinolone resistance genes (pmqr) in P. aeruginosa have also been reported (11,12) and attribute to high levels of resistance albeit at a lower scale compared to mutations in the qrdr region of the bacterium. Frequently, these resistance genes are acquired from other organisms via plasmids, transposons, bacteriophages or integrons (13).
Fluoroquinolones are an important class of wide spectrum antibacterial agents; an example is ciprofloxacin which has emerged as one of the most effective antibiotics against P. aeruginosa (14). Fluoroquinolones target DNA gyrase (topoisomerase II) and topoisomerase IV, which are vital in replication of bacterial DNA. DNA gyrase consists of A2 and B2 subunits encoded by the gyrA and gyrB genes. Topoisomerase IV is encoded by parC and parE subunits (15)(16)(17).
Alterations in QRDR in both gyrA and parC genes are now known to play an integral role in quinolone resistance in P. aeruginosa (18)(19)(20).
The aim of this study was to find possible mutations in gyrA of DNA gyrase and parC of topoisomerase IV in ciprofloxacin-resistant clinical isolates of P. aeruginosa. The correlation between these mutations and minimal inhibitory concentrations (MICs) of ciprofloxacin-resistance was also determined.

MATERIALS AND METHODS
Bacterial isolates. Thirty seven isolates were obtained from patients with burn wounds admitted to Shahid Mottahari Burn Hospital and 20 isolates collected from blood, urine and sputum specimen at Shahid Rajaei Burn Hospital (2009)(2010)(2011)(2012)(2013) in Tehran, Iran. Resistance to ciprofloxacin was evaluated by the Kirby-Bauer test (21). P. aeruginosa ATCC 27853, E. coli ATCC 25922 and S. aureus ATCC 25932 were used as susceptibile controls and compared to CLSI references (22)(23)(24). MIC of ciprofloxacin was measured by microdilution in ciprofloxacin-resistant isolates.
PCR amplification and DNA sequencing. DNA was extracted from ciprofloxacin-resistant isolates by the SET buffer method (25). QRDR amplification of gyrA and parC from resistant isolates was carried out using specific primers: gyrA-1 (5'-GTGTGCTTTATGCCATGAG-3') and gyrA-2 (5'-GGTTTCCTTTTCCAGGTC-3') for the amplification of 287 bp of the fluoroquinolone resistance-determining region of the gyrA gene and parC-1 (5'-CATCGTCTACGCCATGAG-3') and parC-2 (5'-AGCAGCACCTCGGAATAG-3') were used to amplify 267 bp of the fluoroquinolone resistance-determining region of parC as previously reported (10). In the design of parC amplification, the annealing temperature was increased to 59˚C and for some samples up to 60˚C. PCR enhancer was also added to augment the efficiency of PCR (26,27). Amplified products were then separated using 1.5% agarose gels and PCR products were sequenced (Bioron, Germany).
Analysis of DNA sequences. DNA sequences obtained using forward and reverse primers were processed by Bioeditor program using pairwise alignment. The sequence of each of sample was compared with P. aeruginosa PAO1 sequence. The sequences were multiple aligned by Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) in order to detect mutations. Nucleotide Sequences were translated by Expasy Bioinformatics Resource Portal (http://web.expasy.org/translate/) then compared with P. aeruginosa PAO1 protein sequence using Clustal W2 to find changes in amino acids sequences.
Amongst the 57 isolates, 30 isolates were selected  PCR amplification of gyrA and parC genes were carried out using DNA from 22 ciprofloxacin resistant isolates of which 10 were selected for gyrA sequencing and 8 for parC sequencing. Finally, in order to detect the correlation between resistance and gyrA and parC mutations, the results of sequencing were analyzed (Figs. 3 and 4).
Amplification of gyrA resulted in specific bands of 300 bp (Fig. 2). P. aeruginosa strain PAO1 was used as a control for the presence of qrdr region, isolate B48 which was sensitive was used for comparison and a negative control was used as contamination control.

DISCUSSION
Due to the growing number of antibiotic resistant bacteria, the significance of this resistance in MDR P. aeruginosa strains must be taken into greater consideration. The elucidation of the mechanisms http://ijm.tums.ac.ir   leading to this resistance is one of the key factors involved in the treatment of hospital patients with more narrow spectrum, target specific and modified antibiotics (10).
In this study, resistance to ciprofloxacin was 41.37% which is close to the report by Tohidpour and colleagues in Tehran (35%) (28) but lower than the rates reported by Saderi and coworkers' study in Tehran (55%) (29) and another report of Nouri and cowork-ers from Tabriz, Iran (30). In addition, results from the study by Lu and coworkers showed resistance of P. aeruginosa to ciprofloxacin in Asia-Pacific region from 2009 to 2010 was around 44.4% (31).
However, the resistance to ciprofloxacin obtained in this study was higher compared to reports from Canada (27%) (32) and also higher than published data from USA (33.1%) (33).
Statistics indicate that different resistance patterns exist among various regions. The magnitude of antibiotic use might contribute to the variety of antibiotic resistance range; therefore, elevated resistance rate in Asia might be associated with the notably increased use of antibiotics in the area (29,31). One of the major mechanisms involved in the development of quinolone resistance is the mutational alterations in DNA gyrase. In this study, almost 90% of resistant isolates had a gyrA mutation and the most common was the conversion of threonine which is a polar amino acid to non-polar isoleucine amino acid at codon 83. Moreover, high-level resistance is usually associated with the presence of both gyrA and parC mutations simultaneously and one of the frequent mutation in parC changed serine 87 to leucine and more importantly, to tryptophan ( Table  1, Figs 3 and 4). Pasca and coworkers have reported an overwhelming percentage of gyrA and parC mutations as causes for fluoroquinolone resistance in P. aeruginosa isolates from Northern Italian hospitals (34).
Kulberge and her colleagues have shown that single mutations in gyrA or gyrB caused low-level resistance whereas high-level resistant mutants had double mutations in gyrA and parC, parE, nfxB or unknown genes (16).
An evaluation of samples collected in this report from urine, burn wounds and sputum, show high level resistance with MIC>8 μg/ml and mutations in gyrA and parC in the isolates.
In the present study, among 8 resistant isolates, 7 had both gyrA and parC mutations causing relatively high level resistance (MIC≥16 μg/ml). The resistance of 1 isolate (S14) with only silent mutations was comparatively lower than the other resistant mutants (MIC=8 μg/ml and inhibitory zone diameter of 15 mm). It can hence be concluded that other resistance mechanisms except mutations in gyrA and parC can be held responsible for this low-level resistance ( Table 1).
The alteration of polar threonine (Thr) to the nonpolar and highly hydrophobic isoleucine (Ile) does not occur in active site of enzyme; thus the enzyme is able to maintain its function. This mutation is likely to influence the gyrase-quinolone interaction by loss of essential enzyme-drug contacts or conformational modifications that may ultimately result in antibiotic resistance. The presence of gyrA mutation in all resistant isolates endorses the fact that DNA gyrase is pivotal target enzyme in ciprofloxacin resistance in P. aeruginosa (35)(36)(37).
This data substantiates the experiment conducted by Higgins and coworkers in 2003 indicating that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated mainly through mutations in gyrA and mutations in parC genes are subsidiary (38). Reports by Salma and coworkers (39) in Lebanon also substantiate our results and other studies (27,36) that P. aeruginosa resistant mutants with sole parC mutations have not been detected.

CONCLUSION
Results from this study and validation from previous research postulate that gyrA mutations are the major mechanism of resistance to fluoroquinolone for clinical strains of P. aeruginosa and demonstrate that DNA gyrase encoding gene, gyrA is the primary target for fluoroquinolone and further mutations in parC could lead to a higher level of quinolone resistance. This is the first report of parC mutations detected in addition to gyrA mutations reported earlier in resistant P. aeruginosa isolates from Tehran, Iran.